1. Field of the Invention
This invention relates to novel polypeptides which are modifications of fragments of transforming growth factors (TGF). Certain of these compounds are immunogens and thus raise antibodies which are useful for detecting the presence of TGF.alpha. in the samples of fluids. These antibodies can be combined with a conjugate to prepare a diagnostic kit. Certain of the compounds of this invention are analogs of TGF.sub..alpha. fragments or the homologous epidermal growth factor (EGF) fragments which are antagonists of TGF.alpha. and are useful for blocking the cell proliferation effects of TGF.alpha. in a mammal.
2. Related Art
Transforming growth factors (TGF) comprise a family of hormone-like polypeptides which confer the transformed or tumor phenotype on normal cells. Such transformed cells are stimulated to lose anchorage dependence and contact inhibition of growth. The human TGF.alpha. molecule is structurally related to human epidermal growth factor (EGF) and binds to EGF receptors. TGF.alpha. has been detected at elevated levels in the urine of patients with several classes of solid tumors (e.g. bronchogenic carcinoma, breast and bowel carcinoma, and the like). The development of an assay and diagnostic kit capable of detecting and quantitating the TGF.alpha. molecule without excess cross-reactivity with EGF would be of great interest in diagnosing and managing many classes of solid tumors.
The most convenient and sensitive way to detect TGF.alpha. would be through the use of a specific antibody-based assay system. However, the antibodies would have to be raised against TGF.alpha., and TGF.alpha. would have to be used as a calibration reagent to accompany the assay system. Because the purification and isolation of TGF.alpha. is difficult and expensive, the use of TGF.alpha. for these purposes is not economically feasible at the present time.
The structure of rat transforming growth factor has been reported to be Val-Val-Ser-His-Phe-Asn-Lys-Cys-Pro-Asp.sup.(10) -Ser-His-Thr-Gln-Tyr-Cys-Phe-His-Gly-Thr.sup.(20) -Cys-Arg-Phe-Leu-Val-Gln-Glu-Glu-Lys-Pro.sup.(30) -Ala-Cys-Val-Cys-His-Ser-Gly-Tyr-Val-Gly.sup.(40) -Val-Arg-Cys-Glu-His-Ala-Asp-Leu-Leu-Ala [Marquardt, et al., Science, 223, 1079 (1984)]. The human TGF.alpha. sequence is closely related and, in residues 34-43 differs from the rat TGF only by the replacement of the valyl residue by an alanyl residue at position 41 [Derynck, et al., Cell, 38, 287 (1984)]. Human EGF is reported to be a 53 residue polypeptide with the sequence of residues 34-43 being Cys-Val-Val-Gly-Tyr-Ile-Gly-Glu-Arg-Cys [Carpenter and Cohen, Ann. Rev. Biochem., 48, 193-216 (1979)].
The use of a suitable polypeptide fragment, instead of the whole TGF molecule, to raise antibodies and as a calibration reagent would offer the advantages of easier synthesis and lower cost.
We have discovered that certain polypeptides, which are modified fragments of TGF.alpha., produce specific antibodies which react with TGF.alpha. but do not cross-react with EGF, thus providing a means for a diagnostic system which is capable of detecting TGF.alpha. in the presence of EGF. We have also discovered that polypeptides, which are modified fragments of TGF.alpha., bind to EGF receptors but do not stimulate epidermal cell growth. Thus, these compounds are competitive inhibitors of TGF.alpha. and as such are useful for blocking the cell proliferation effects of TGF.alpha.. In addition, the corresponding fragment of EGF and analogs thereof bind to the EGF receptor and also represent antagonists of EGF or TGF.